Abstract

Genetic mutations in muscle structural genes can compromise myofiber integrity, causing repeated muscle damage that ultimately exhausts muscle regenerative capacity and results in devastating degenerative conditions such as Duchenne Muscular Dystrophy (DMD), Congenital Muscular Dystrophy (CMD) and different forms of Limb Girdle Muscular Dystrophy (LGMD). Gene supplementation and autologous stem cell transplant have been put forward as promising, though still unproven, therapeutic avenues for combatting these genetic muscle diseases. Both strategies aim to compensate expression of the missing or mutated protein. For cell therapy, autologous muscle stem cells (satellite cells) from dystrophic muscles undergo in vitro expansion and gene correction and then are transplanted into diseased tissue, where they fuse with resident myofibers to deliver a functional copy of the gene. One of the major obstacles for the autologous adult stem cell transplantation is that adult satellite cells account for a very rare population in muscle and they need to be expanded in culture, while retaining their engraftment potential, to generate sufficient number of cells for gene correction and transplantation. I tackled this problem by developing a culture condition that allows engraftable mouse satellite cells to expand in culture. This study also provides evidence for the feasibility of in vitro expansion, gene correction and transplantation of dystrophic satellite cells to restore DYSTROPHIN expression in dystrophic muscle.In gene therapy, engineered gene products are delivered directly to muscle fibers as transgenes carried by viral vectors, such as Adeno Associated Viruses (AAVs). Viral- mediated delivery of a normal copy of the mutated genes into dystrophic muscle fibers holds big promise as a therapeutic avenue for Muscular Dystrophies. However, considering the indispensible role of satellite cells in muscle regeneration, an effective and long-term therapy for genetic muscle diseases requires restoration of gene expression in both dystrophic muscle fibers and satellite cells. Conventional gene therapy approaches lack the potential for long-term restoration of the mutated gene expression in satellite cells. In order to address this limitation, this study provides the proof of concept evidence for the use of a novel gene editing approach, which allows irreversible correction of the mutations in both dystrophic skeletal muscle fibers and satellite cells.